L-proline has been postulated to be a neurotransmitter or neuromodulator in the mammalian central nervous system (CNS). Studies to define the role of L-proline in discrete pathways in the mammalian CNS have not been possible due to the inability to block its biosynthesis or high affinity transport in nervous tissue. Our recent cloning of a high affinity, Na+- dependent L-proline transporter expressed in subpopulations of putative glutamatergic neurons provides compelling support for a specific role for L-proline in excitatory transmission. Glutamatergic synapses have been implicated in diverse physiologic processes including the acquisition of associative learning, synapse formation, and neuroendocrine regulation. Abnormalities of glutamatergic transmission have been implicated in the pathophysiology of excitotoxic neurologic disorders. Thus an understanding of the functional role(s) of the L-proline transporter in specific glutamatergic neurons could provide insights into synaptic regulatory mechanisms involved in synaptic plasticity and memory acquisition or their disruption in diseases such as Alzheimer's disease. The long term goal of this research is to elucidate the role(s) of L-proline, and its transporter, in synaptic transmission. Specific Aim l will undertake the first detailed studies of the regional, cellular, and subcellular distribution of the high affinity L-proline transporter protein in rat brain. The precise projection pathways of individual neurons that express L-proline transporter mRNA will be determined by combining retrograde dye tract-tracing with in situ hybridization histochemistry. Knowledge of these pathways will be crucial to the design of physiological studies of the role of L-proline in synaptic transmission. Studies will also examine the possible co-localization of high affinity L-proline and L-glutamate transporters in glutamatergic nerve terminals. Specific Aim 2 will use the L-proline transporter as a model neurotransmitter transporter, to investigate the structure-function properties and regulation of this important class of synaptic proteins using chimeric transporter strategies and site-directed mutagenesis. The final Specific Aim will investigate potential synaptic roles of L-proline. A Xenopus oocyte expression assay will be used to investigate the hypothesis that L-proline modulates signal transduction at a glutamate receptor subtype, analogous to glycine modulation of the NMDA receptor. This assay will also be used to investigate the possibility that distinct L-proline receptors exist in mammalian CNS. Monoclonal antibodies that selectively block L-proline transport will be generated, for use as transport inhibitors, to examine the hypothesis that L-proline modulates the synthesis and/or release of glutamate from nerve terminals. The potential discovery of novel regulatory mechanisms in glutamatergic synapses is the incentive which drives the research described in this proposal.